1. Field of the Invention
This invention relates to a method of preparing an integral multilayer analytical element for analysis of an analyte in a liquid sample. More particularly, this invention relates to an improvement of a method of preparing a dry-type integral multilayer analytical element for analysis of an analyte in a liquid sample, such as, a biological body fluid, including blood (whole blood, blood plasma, blood serum), cerebrospinal fluid, lymph, saliva and urine, and which is useful for diagnosis in the clinical field.
2. Description of the Prior Art
Various integral multilayer analytical elements have been known. However, in every analytical element, when a water-soluble substance is used as the indicator for reacting with the analyte in a sample to generate an optical change, the indicator diffuses into the upper porous spreading layer and results in a lowering of the analytical accuracy. (This diffusion is called "migration".) In the case that the indicator is highly soluble in water, such as in the case of the analytical element for analysis of calcium, the analytical accuracy is remarkably lowered by the migration. Since the change in the calcium concentration in blood does not vary widely, this defect is fatal for this analytical element.
Therefore, various investigations have been made in order to eliminate the defect. For example, a migration-inhibiting layer can be introduced into the analytical element as disclosed in U.S. Pat. No. 4,166,093. This migration-inhibiting layer inhibits the migration of a water-soluble indicator by immobilizing it with a polymer mordant. However, the mordant often inhibits coloration of the indicator and, accordingly, it is not a fundamental solution to this problem. Besides, the preparation of this layer is difficult, thereby raising the cost of the analytical element.
It is also a problem that an aqueous liquid sample spotted on a porous spreading layer spreads too broadly in this spreading layer. In EP 0,l62,302A, hydrophilic cellulose derivatives and nonionic surfactants having an HLB value of more than 10 are disclosed as being effective as a spreading action controller. The hydrophilic cellulose derivative is dissolved in water and applied on the spreading layer. Since the solubility of the nonionic surfactant in water is low, it is dissolved in a mixture solvent of water and acetone.
On the other hand, colorimetry using an indicator is widely employed in the field of clinical analysis. The indicator is usually o-Cresolphthalein Complexone of which the optimum pH for coloration is in the alkaline range, particularly higher than pH 10 where the binding to calcium is stable. Also, it is known that the samples for clinical diagnosis, such as, blood, contain the calcium in a bound state, such as, protein-bound calcium and acid-bound calcium, in addition to the calcium in an ion state. All calcium in the sample is first ionized by an acid treatment, and thereafter, a color reaction is carried out (Clin. Chem. Vol. 29, p. 1497 ( 1983)). This method is, for example, described on pages 148 to 150 of "Jissen Rinsho Kagaku-Zoho Ban (Practical Clinical Chemistry-Revised and Enlarged Edition)" (Kitamura et al., Ishiyaku Shuppan Kabushiki Kaisha, Japan 1982). An outline of this method is as follows. o-Cresolphthalein Complexone is dissolved in a small amount IN KOH, and glacial acetic acid is added to this solution to prepare an acidic solution 1. An alkaline solution 2 containing potassium acetate-HCl-diethylamine is separately prepared. The solution 1 is added to a sample, and allowed to react. Subsequently, the solution 2 is added to the reaction mixture, and color reaction proceeds. Recently, a one-step type reagent kit has also been available ("Agent Calcium", OINABBAT, U.S.A.). It has been reported that a linear relation between absorbance and concentration is obtained by allowing citric acid, its salt, or mixtures thereof, to coexist throughout the reaction (Japanese Patent Kokai No. 57-154058). However, in this patent, citric acid is used in a concentration of 0.01 to 0.05 M/l in an alkaline condition, such as, pH 10 to 11, but this acid is not utilized for acidification of the sample nor the color reaction.
In the case of the analytical element for analysis of calcium, in order to decompose the bound state of calcium, the pH of the upper layer is made lower than 5, usually pH 1 to 2. On the other hand, the optimum reaction pH of preferable indicators, such as, o-Cresolphthalein is usually higher than pH 10. Since it is difficult for such a high pH layer to coexist with the low pH layer in a thin analytical element, the coloring reaction was set to proceed at about pH 5.5. Chlorophosphonazo-III and Arsenazo-IIl were used as the indicator. However, in this analytical element, the absorbance of the background is high, such as, higher than 2 and the measurement is carried out at a long wave length (near 680 nm). Accordingly, the analytical accuracy becomes worse.